A complete Protocadherin-19 ectodomain model for evaluating epilepsy-causing mutations and potential protein interaction sites

Hudson, J.D.; Tamilselvan, Elakkiya; Sotomayor, Marcos; Cooper, Sharon

doi:10.1016/j.str.2021.07.006

Cited by 7 publications

(7 citation statements)

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“…To model mild skewing of RXI that is likely to occur in patients, we next generated PCE organoids with different ratios of WT:KO cells (1:1, 2:1, 1:2) and found significant cell segregation from all mixing conditions in comparison to WT only and KO only cultures (Supplementary Figures S6, S8), with no quantitative difference in striping between the three mosaic conditions (Supplementary Figure S6B, adjusted P > 0.999). In order to validate that our PCE organoid model contains mosaic expression of PCDH19, we immunostained day 20 WT, KO and WT/KO (PCE) hCOs with anti-HA antibody to identify PCDH19 protein, as well as N-cadherin (NCAD), a PCDH19 interacting protein (Cooper et al, 2016;Hoshina et al, 2021;Hudson et al, 2021). We confirmed that PCDH19 localizes to the apical junction in all WT organoids, and displays distinct apicalbasal patterns (Figures 3D, 4A).…”

Section: Pce Hco Models Show a Cell Sorting Phenotypementioning

confidence: 57%

“…In order to validate that our PCE organoid model contains mosaic expression of PCDH19, we immunostained day 20 WT, KO and WT/KO (PCE) hCOs with anti-HA antibody to identify PCDH19 protein, as well as N-cadherin (NCAD), a PCDH19 interacting protein ( Cooper et al, 2016 ; Hoshina et al, 2021 ; Hudson et al, 2021 ). We confirmed that PCDH19 localizes to the apical junction in all WT organoids, and displays distinct apical-basal patterns ( Figures 3D , 4A ).…”

Section: Resultsmentioning

confidence: 99%

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Abnormal cell sorting and altered early neurogenesis in a human cortical organoid model of Protocadherin-19 clustering epilepsy

Niu,

Deng,

Mojica-Perez

et al. 2024

Front. Cell. Neurosci.

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IntroductionProtocadherin-19 (PCDH19)-Clustering Epilepsy (PCE) is a developmental and epileptic encephalopathy caused by loss-of-function variants of the PCDH19 gene on the X-chromosome. PCE affects females and mosaic males while male carriers are largely spared. Mosaic expression of the cell adhesion molecule PCDH19 due to random X-chromosome inactivation is thought to impair cell–cell interactions between mutant and wild type PCDH19-expressing cells to produce the disease. Progress has been made in understanding PCE using rodent models or patient induced pluripotent stem cells (iPSCs). However, rodents do not faithfully model key aspects of human brain development, and patient iPSC models are limited by issues with random X-chromosome inactivation.MethodsTo overcome these challenges and model mosaic PCDH19 expression in vitro, we generated isogenic female human embryonic stem cells with either HA-FLAG-tagged PCDH19 (WT) or homozygous PCDH19 knockout (KO) using genome editing. We then mixed GFP-labeled WT and RFP-labeled KO cells and generated human cortical organoids (hCOs).ResultsWe found that PCDH19 is highly expressed in early (days 20–35) WT neural rosettes where it co-localizes with N-Cadherin in ventricular zone (VZ)-like regions. Mosaic PCE hCOs displayed abnormal cell sorting in the VZ with KO and WT cells completely segregated. This segregation remained robust when WT:KO cells were mixed at 2:1 or 1:2 ratios. PCE hCOs also exhibited altered expression of PCDH19 (in WT cells) and N-Cadherin, and abnormal deep layer neurogenesis. None of these abnormalities were observed in hCOs generated by mixing only WT or only KO (modeling male carrier) cells.DiscussionOur results using the mosaic PCE hCO model suggest that PCDH19 plays a critical role in human VZ radial glial organization and early cortical development. This model should offer a key platform for exploring mechanisms underlying PCE-related cortical hyperexcitability and testing of potential precision therapies.

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Section: Pce Hco Models Show a Cell Sorting Phenotypementioning

confidence: 57%

Section: Resultsmentioning

confidence: 99%

Abnormal cell sorting and altered early neurogenesis in a human cortical organoid model of Protocadherin-19 clustering epilepsy

Niu,

Deng,

Mojica-Perez

et al. 2024

Front. Cell. Neurosci.

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“…This deletion caused a frameshift mutation, resulting in the substitution of the 99 th threonine with serine and the appearance of a new reading frame. Translation terminated at the 8 th codon downstream (Figure 1C )[ 25 ]. Protein tertiary structure prediction showed that the normal MERTK protein exhibited complex protein domains, while the mutant MERTK protein retained only a short peptide chain (Figure 1D ).…”

Section: Resultsmentioning

confidence: 99%

Patient-derived induced pluripotent stem cells with a MERTK mutation exhibit cell junction abnormalities and aberrant cellular differentiation potential

Zhang,

Wu,

Liu

et al. 2024

World J Stem Cells

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BACKGROUND Human induced pluripotent stem cell (hiPSC) technology is a valuable tool for generating patient-specific stem cells, facilitating disease modeling, and investigating disease mechanisms. However, iPSCs carrying specific mutations may limit their clinical applications due to certain inherent characteristics. AIM To investigate the impact of MERTK mutations on hiPSCs and determine whether hiPSC-derived extracellular vesicles (EVs) influence anomalous cell junction and differentiation potential. METHODS We employed a non-integrating reprogramming technique to generate peripheral blood-derived hiPSCs with and hiPSCs without a MERTK mutation. Chromosomal karyotype analysis, flow cytometry, and immunofluorescent staining were utilized for hiPSC identification. Transcriptomics and proteomics were employed to elucidate the expression patterns associated with cell junction abnormalities and cellular differentiation potential. Additionally, EVs were isolated from the supernatant, and their RNA and protein cargos were examined to investigate the involvement of hiPSC-derived EVs in stem cell junction and differentiation. RESULTS The generated hiPSCs, both with and without a MERTK mutation, exhibited normal karyotype and expressed pluripotency markers; however, hiPSCs with a MERTK mutation demonstrated anomalous adhesion capability and differentiation potential, as confirmed by transcriptomic and proteomic profiling. Furthermore, hiPSC-derived EVs were involved in various biological processes, including cell junction and differentiation. CONCLUSION HiPSCs with a MERTK mutation displayed altered junction characteristics and aberrant differentiation potential. Furthermore, hiPSC-derived EVs played a regulatory role in various biological processes, including cell junction and differentiation.

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“…Despite the γC4 trans dimer sharing structural similarity and the interface having similar buried surface area as alternate α, β, γA, and γB cPcdhs and δ2 nonclustered Pcdhs ( Figure 3—source data 2 ; Cooper et al, 2016 ; Goodman et al, 2016a ; Goodman et al, 2016c ; Harrison et al, 2020 ; Hudson et al, 2021 ; Nicoludis et al, 2016 ), its binding affinity is very weak. The two most structurally similar molecules to γC4 over their trans -interacting domains are cPcdh γB2 and nonclustered Pcdh19.…”

Section: Resultsmentioning

confidence: 99%

How clustered protocadherin binding specificity is tuned for neuronal self-/nonself-recognition

Goodman

Katsamba

Rubinstein

et al. 2022

eLife

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The stochastic expression of fewer than 60 clustered protocadherin (cPcdh) isoforms provides diverse identities to individual vertebrate neurons and a molecular basis for self-/nonself-discrimination. cPcdhs form chains mediated by alternating cis and trans interactions between apposed membranes, which has been suggested to signal self-recognition. Such a mechanism requires that cPcdh cis dimers form promiscuously to generate diverse recognition units, and that trans interactions have precise specificity so that isoform mismatches terminate chain growth. However, the extent to which cPcdh interactions fulfill these requirements has not been definitively demonstrated. Here, we report biophysical experiments showing that cPcdh cis interactions are promiscuous, but with preferences favoring formation of heterologous cis dimers. Trans homophilic interactions are remarkably precise, with no evidence for heterophilic interactions between different isoforms. A new C-type cPcdh crystal structure and mutagenesis data help to explain these observations. Overall, the interaction characteristics we report for cPcdhs help explain their function in neuronal self-/nonself-discrimination.

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A complete Protocadherin-19 ectodomain model for evaluating epilepsy-causing mutations and potential protein interaction sites

Cited by 7 publications

References 80 publications

Abnormal cell sorting and altered early neurogenesis in a human cortical organoid model of Protocadherin-19 clustering epilepsy

Abnormal cell sorting and altered early neurogenesis in a human cortical organoid model of Protocadherin-19 clustering epilepsy

Patient-derived induced pluripotent stem cells with a MERTK mutation exhibit cell junction abnormalities and aberrant cellular differentiation potential

How clustered protocadherin binding specificity is tuned for neuronal self-/nonself-recognition

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